The present invention relates to novel biologically active compounds from fungi of the species Ganoderma pfeifferi DSM 13239, processes for their preparation, and their use. From the fruit body and mycelium of the species Ganoderma pfeifferi DSM 13239, extracts can be obtained which have antimicrobial activity and are suitable as preserving agents, for pharmaceutical and cosmetic preparations, for controlling infections, and for use in fish breeding.
Biologically active compounds which can be obtained from certain fungi of the genus Ganoderma by extraction with solvents have long been described and summarized by us in a monograph (U. Lindequist: Ganoderma. In: Hagers Handbuch der pharmazeutischen Praxis/Ed. F. von Bruchhausen, 5th Edition, totally revised, Springer Publishers Berlin, Heidelberg, N.Y., 1998, Supplement Volume 2, Drogen A-K (Ed. W. Blaschek), pages 750-761. In the following, reference is made to the literature stated therein.
To date, commercially utilized drug-supplying species of the genus Ganoderma only include Ganoderma applanatum (Ganoderma applanatum fruit bodies) and Ganoderma lucidum (Ganoderma lucidum fruit bodies). Due to its valuable components, G. lucidum is grown commercially in various Asian countries (Japan, China, Korea etc.) in large amounts on artificial substrate (Hager, references 20, 25).
For extracting the active substances from G. lucidum and G. applanatum, various solvents have been employed. To date, dichloromethane and ethyl acetate have not been used.
The most important group of active substances of these fungi are the triterpenes, polysaccharides and sterols. More than 100 triterpenes have been structurally elucidated. They are referred to, inter alia, as ganoderic acids, ganodermic acids, ganoderenic acids, ganolucidic acids, ganosporeric acids, lucidenic acids, ganoderiols, epoxyganoderiols, ganoderals, ganoderols, lucidones, ganodermanonediol, ganodermanonetriol and ganodermatriol. Due to the different designations of a compound in different working groups, there are numerous double usages and unclarities. The basic body is a lanostane, which is mostly unsaturated, often with two conjugated double bonds in the ring system in positions 7 and 9(11), or one double bond in position 8 which is in conjugation with two oxo groups in positions 7 and 11. The acids have a carboxy group at the end of the side chain in position 26. In the trinortriterpenic acids (C27 compounds), the lucidenic acids, the carboxy group is in position 24. Methyl esters of the acids also exist. The hydroxy groups are in part acetylated or dehydrogenated into oxo groups. In some members, there has been degradation of the side chain to as few as two carbon atoms (C23 compounds).
During the development of the fruit bodies, changes in the triterpene pattern of the fungi occur. In the mycelium stage, the ganoderic acids are predominant, but with progressing development of the fruit bodies, lucidenic acids are increasingly occurring (Hager, reference 22). In the polysaccharides, a distinction can be made between glucanes, heteropolysaccharides and protein-bound polysaccharides.
In addition, various proteins, steroids, nucleotides and some further small-molecular compounds have been characterized in Ganoderma lucidum. 
As the main sterols, ergosterol and ergosta-7,22-dienol have been identified (Hager, references 15, 56, 65). In addition, ergosterol peroxide, ergosta-7,22-diene-3xcex2-yl palmitate (56, 66), 6xcex1- and 6xcex2-hydroxyergosta-4,7,22-triene-3-one (52), ergosta-7,22-diene-3xcex2-yl linoleate, 5xcex1,8xcex1-epidioxyergosta-6,22-diene-3xcex2-yl linoleate and ergosta-7,22-diene-2xcex2-3xcex1,9xcex1-triol (66) have been identified.
Further components are adenosine in concentrations of from 40 mg/100 g of dried fruit bodies (Hager, reference 67), and 5xe2x80x2-deoxy-5xe2x80x2methylsulfinyladenosine (Hager, reference 68).
From extracts of the cultured mycelium, a polypeptide referred to as Ling Zhi-8 (LZ-8) and consisting of 110 amino acids and having a molecular weight of 12,420 Dalton has been isolated whose amino end is acetylated (Hager, references 69, 70). In addition, a lectin consisting of 2 protein subunits (molecular weights 55,600 and 59,800 Dalton) and a carbohydrate content of 2.56% has been found (Hager, reference 72).
Extracts from G. lucidum have long been used as medicaments. The effects of extracts from Ganoderma lucidum are very complex and varied, and it was seldom possible to date to assign them unambiguously to individual active substances. The following effects of extracts from G. lucidum belong to the prior art.
Extracts having effects on the CNS: An aqueous extract of the fruit bodies has centrally inhibiting and muscle-relaxing effects in mice. In a dose-dependent way, 30, 100 and 300 mg of the lyophilized extract per kg of body weight, s.c., suppress the activity of the animals in a running wheel, the locomotive activity and the centrally stimulating effect of caffeine. The pain threshold in the heat plate test is increased. In the tail pressing test, analgesic effects occur. 300 mg/kg prolongs the time until onset of death after the administration of strychnine (1.5 mg/kg, i.p.) or caffeine (400 mg/kg, i.p.), the spasms induced by the alkaloids not being influenced (Hager, reference 73). In rats, Ganoderma extracts are said to have a sleep-promoting effect (Hager, reference 74).
Extracts having effects on the cardiovascular system: Hexane and methanol extracts of the fruit bodies (1 mg/ml) significantly reduce the contraction rate of cultivated myocardial cells of mice in vitro by 25 and 80%, respectively. The hexane and aqueous extracts increase the contraction amplitude by about 15% at the mentioned concentration. Responsible compounds for the former effect are ganoderic acid S, ganoderal A and ganodermanonetriol. 0.1 mg/ml of this compound will result in a complete stop of the contractions. The increase of the amplitude is caused by ganoderic acid S, portensterol and ganodermanonetriol (Hager, reference 5).
In vivo, a lyophilized aqueous extract was administered to patients with essential hypertension (group I) and to patients with a mild or no hypertension (group II) orally in the form of tablets over a period of 6 months (6 tablets with 240 mg of extract per day). In the patients of group I, a significant decrease of the increased blood pressure occurred while that of patients of group II was not influenced. Side-effects did not occur (Hager, reference 76).
Spontaneously hypertensive rats whose feed contained 5% Ganoderma lucidum powder exhibited a significantly reduced systolic blood pressure and a reduced total cholesterol level in the plasma and liver as compared to the control group after 4 weeks with a corresponding diet (Hager, reference 77).
Morigiwa et al. (Hager, reference 39) found inhibition of the angiotensin-converting enzyme in 10 triterpenes in vitro and thus another possible explanation of the antihypertensive effects. The most potent was ganoderic acid F (IC50 4.7xc3x9710xe2x88x926 M). Ganoderal A, ganoderols A and B and the ganoderic acids B, D (C), H, K, S and Y were active with an average IC50 of 10xe2x88x925 M.
Extracts having effects on the blood: An aqueous extract (50 xcexcl of the extract, 5 g of the fungus extracted with 300 ml) inhibits the thrombin-induced aggregation of bovine platelets in vitro. Adenosine has been identified as the responsible active substance (Hager, reference 67). On the other hand, raw extracts do not exhibit an anti-platelet activity in HIV-positive hemophilic patients despite of high adenosine concentrations (Hager, reference 78). Further active substances for which an effect on platelet aggregation has been detected in vitro include 5-deoxy-5-methylsulfinyladenosine (Hager, reference 79) and ganodermic acid S. In a concentration of 50 xcexcg/ml, the adenosine derivate inhibits the ADP-induced aggregation of rabbit platelets, but not the PAF-stimulated aggregation (Hager, reference 79). Ganodermic acid S has membrane activity and influences the phosphoinositol metabolism in vitro. Depending on its concentration, it promotes or inhibits the aggregation of human platelets caused by different inductors in vitro (Hager, references 80, 81).
In an animal experiment, an aqueous extract (500 mg/kg) inhibits the decrease of platelet and fibrinogen levels and the prolongation of the prothrombin time in rats in which disseminated intravascular coagulations had been produced by the action of endotoxins. In vitro, this extract in concentrations of from 500 to 1000 xcexcg/ml reduces the action of thrombin and the collagen-induced platelet aggregation (Hager, reference 82).
In addition, the influence of aqueous extracts on the aggregation of platelets from 15 healthy donors and 33 patients with arteriosclerosis was examined. In-vitro addition of the extract to the platelets of healthy subjects reduces aggregation in a concentration-dependent way. In vivo, the oral administration of 1 g of extract three times a day for 2 weeks to the patients results in a significant inhibition of ADP-induced platelet aggregation (Hager, reference 83).
Extracts having effects on the digestive tract: Some triterpenes (lucidenic acid A, lucidenic acid D1, ganoderic acid A, ganoderic acid C1, ganoderic acid J, lucidone A, lucidone C) have an intensive bitter taste and thus should have a digestion-stimulating and appetizing effect. The bitter taste of lucidenic acid D1 is still discernible in a dilution of 5xc3x9710xe2x88x9210 (Hager, reference 84).
Extracts having effects on the liver: In vitro, a strong inhibition of the division of human hepatoma cells (PLC/PRF/5 cells) and of KB cells by ganoderaldehyde A (ED50 for the hepatoma cells: 10.99 xcexcg/ml; for KB cells: 9.75 xcexcg/ml) and by ergosta-7,22-diene-2xcex2,3xcex1,9xcex1-triol (ED50 for the hepatoma cells: 1.17 mg/ml; for KB cells: 0.89 xcexcg/ml) was achieved (Hager, reference 66).
The ganoderic acids T, U, V, W, X, Z also exhibit cytotoxic activity on hepatoma cells in vitro in doses of 10xe2x88x924 mol (Hager, reference 47).
In contrast, the ganoderic acids R and S have an antihepatotoxic effect in vitro. They reduce the galactosamine-induced cytotoxicity in cultured rat hepatocytes (Hager, reference 45).
In rats, aqueous extracts of the fruit bodies (30 and 100 mg/kg, i.p.) reduce chloroform-induced liver toxicity (determination of GOT and LDH) (Hager, reference 85). In mice, alcoholic extracts antagonize the accumulation of lipid in the liver, which is also caused by chloroform. In addition, they reduce the mortality rate of the animals upon high doses of digitoxin and indomethacin. They promote the liver regeneration in partially hepatectomized animals (Hager, reference 6).
Two polysaccharide fractions obtainable from the mycelium by alkali treatment and ethanol precipitation (0.5 to 5 mg/rat/day for 4 weeks) exhibit antifibrotic effects in rats having experimentally induced hepatic cirrhosis (Hager, reference 87).
Extracts having effects on metabolism: Aqueous and ethanolic extracts of the fruit bodies were tested for their influence on the glucose and insulin levels in an oral glucose tolerance test in rats. The aqueous extract (50 mg/rat weighing 200-250 g) significantly reduced the glucose level in the blood after 10 min from an oral glucose administration as compared to the control rats. After 10 min, the insulin level was also reduced as compared to the control group, but 30 and 60 min after the glucose supply, it was on a significantly higher level as compared to the control group. In addition, the aqueous extract reduced the increase of the blood glucose level induced by i.v. administration of adrenalin and the adrenalin-induced lipolysis in rat adipocytes. The glucose absorption from the small intestine was not influenced. It is supposed that the effect of the aqueous extract is based on an improvement of glucose utilization in the peripheral tissue. The ethanolic extracts were hardly effective (Hager, reference 88).
Hikino et al. (Hager, references 62, 63, 89) found hypoglycemic effects in the polysaccharides referred to as ganoderanes. There is no overlap with the polysaccharides having antitumor activity (Hager, reference 90). The most active compound is ganoderane B. After 3 to 7 hours from the i.p. administration, 30 mg/kg of this compound significantly reduce the plasma glucose level in glucose-loaded mice. In normal animals, the effect is not so clearly pronounced. The hypoglycemic activity is attributed to an increase of the insulin concentration in the blood plasma and an enhancement of glucose metabolism (Hager, reference 63).
Other metabolic effects relate to the cholesterol level in the blood. Aqueous and alcoholic extracts of Ganoderma lucidum inhibit the cholesterol accumulation in cultured human aortic intimal cells and the proliferation of the cells. Therefore, antiarteriosclerotic properties of the fungus are assumed (Hager, reference 91).
Some oxygenated triterpenes having a carboxy group on C-26 (3xcex1,15xcex1-diacetoxy-5xcex1-lanosta-7,9(11),24-triene-26-carboxylic acid, 3xcex1,15xcex1-dihydroxy-5xcex1-lanosta-7,9(11),24-triene-26-onecarboxylic acid) are hardly absorbed in the gastro-intestinal tract of rats. In this way, much like sitosterol, they reduce the absorption of food-supplied cholesterol from the gastrointestinal tract and thus have a hypolipidemic effect (Hager, references 92, 93). Ganoderic acid derivatives changed by partial synthesis and having 7-oxo and 15xcex1-hydroxy groups inhibit 14-demethylation during the biosynthesis of cholesterol from 24,25-dihydrolanosterol and are also said to be capable of reducing the cholesterol level in the blood (Hager, reference 94).
Extracts having effects on the immune system: Aqueous extracts of the fruit bodies exhibit antiallergic effects. In concentrations of from 20 to 500 xcexcg/ml, they inhibit the release of histamine from peritoneal rat mast cells induced by the compound 48/80 (10 xcexcg/ml) by 14 to 70% (95). In vivo, they reduce symptoms of experimental asthma in Guinea pigs (disorders of the respiratory rate and of the ratio of expiration/inspiration), of contact dermatitis induced by picryl chloride in mice (swelling of the ear), and of serum nephritis caused by immune complexes in rats (protein excretion, blood pressure, microscopic changes in the glomeruli). The dose administered p.o. was 500 mg/kg each (96).
The ganoderic acids C and D isolated from a methanolic extract of the fruit bodies, like the ethyl acetate phase of the starting extract, inhibit the release of histamine from peritoneal mast cells of rats induced by concanavaline A or the compound 48/80 in vitro. At 2 mg/ml, the mentioned compounds reduce the Con-A-induced release by 85% and 80%, respectively, and the 48/80-induced release by 70 and 80%, respectively (Hager, reference 31).
An extract exclusively prepared from the spores (water-soluble fraction of the ethanolic extract of the spores), in doses of 50, 100 and 200 mg/kg/day on 9 days i.p., inhibits the DTH response in mice induced by sheep erythrocytes, 2,4-dinitrochlorobenzene or allotypic splenocytes (Hager, reference 97).
The protein Ling Zhi-8 isolated from the mycelium has a mitogenic in vitro effect on T lymphocytes (Hager, reference 98). In doses of 6.9 mg/kg twice a week i.p., it inhibits the anaphylactic responses induced by bovine serum albumin in mice (99).
A chloroform extract from G. lucidum also exhibits an antiallergic effect (JP 0630 280 028 AA).
Extracts having anti-tumor activity: Extracts from G. lucidum and the ganoderic acids A-Z isolated therefrom exhibit anti-tumor activity (JP 0040304890 AA). Upon i.p. administration, a lyophilized hot-water extract of the fruit bodies alone or in combination with cytostatic agents (adriamycin, methotrexate and others) significantly prolongs the survival time of syngenic C57BL/6 mice into which a Lewis lung carcinoma had been grafted on an i.p. route. Pretreating the mice with cyclosporin inhibited this activity. Cytotoxicity could not be detected. Doses of 10 mg/kg i.p. on days 1, 3, 5, 7 and 9 after the tumor cell inoculation (day 0) prolonged the survival time by 95%. When combined with cytostatic agents, the effects were even more pronounced in part. The activity is attributed to a stimulation of the immune system by polysaccharides contained in the extract (Hager, reference 100). At a dosage of 10 mg/kg i.p. for 10 days, a hot-water extract of the fruit bodies reduces the weight of sarcoma 180 tumors in mice (ICR) by 99%. In one out of three animals, a complete regression occurred. The responsible fractions were those having a molecular weight of more than 10,000 Dalton. In contrast to aqueous extracts, methanolic extracts were not effective upon i.p. or p.o. administration (Hager, reference 101). The growth of a fibrosarcoma in C3H mice is also inhibited by aqueous extracts (Hager, references 102, 103).
Upon i.p. administration in doses of from 5 to 100 mg/kg, polysaccharide fractions obtainable from the fruit bodies by extraction with various solvents (hot water, 3% ammonium oxalate solution, 100xc2x0 C., 5% NaOH, 30xc2x0 C. and 80xc2x0 C., etc.) reduce the growth of s.c. implanted sarcoma 180 tumors in ICR/JCL mice (Hager, references 57, 58, 59). Ten days of i.p. administration of 20 mg/kg of a water-soluble polysaccharide will inhibit the growth of solid sarcoma 180 tumors in mice by more than 90%. As the structure essential to this activity, a glucane with xcex21-3, xcex21-4, and xcex2-1-6 linkages was identified (Hager, reference 102). In doses of 10 mg/kg/day i.p., a glucane fraction isolated in 1985 by Sone et al. resulted in complete regression of sarcoma 180 tumors in four out of five mice after 10 days. The anti-tumor activity is higher with the higher-branched glucanes (Hager, reference 61). A water-soluble polysaccharide fraction inhibited the tumor growth in BDF1 mice with leucemia P388 or L1210 and prolonged the survival time of the animals (Hager, reference 103).
Upon i.p. administration (20 mg/kg/day) to ICR mice, the polysaccharide-protein complex lucidane inhibits the growth of sarcoma 180 tumors by about 70% (64).
From the culture filtrate, an alkali-soluble glucane (1-3-linked glucose units with 1-2 and 1-6 branches) was extracted which inhibits the growth of sarcoma 180 tumors in mice by more than 90% upon i.p. administration in doses of 10 mg/kg/day on 10 days (Hager, reference 61).
After 5 days of repeated p.o. administration of polysaccharides to mice bearing sarcomas 180, a permanent increase of the phagocytosis index occurs (Hager, reference 105). The formation of oxygen radicals in isolated macrophages is stimulated (Hager, reference 103).
In a mixed lymphocyte culture, polysaccharides from Ganoderma lucidum extracts promote the IL-2 production 12 hours after initiation of the culture in a concentration-dependent way and enhance the regeneration of Lyt2+ and L3T4+ cells after 4 days (Hager, reference 107); other extracts have a scavenging effect on free radicals and thus exhibit anti-oxidative effects (Hager, references 85, 109) and cause a delay of ageing processes (Hager, references 60, 112). Promotion of the proliferation and differentiation of a human leucemic monocyte cell line (U 937) into mature monocytes/macrophages by 50 xcexcg/ml of polysaccharides after 24 hours of incubation has been described (Hager, reference 111); doses of 25 and 50 mg/kg/day of a polysaccharide fraction i.p. for 4 days restore the activity of DNA polymerase xcex1 in 24-month-old mice to higher than the activity level of the enzyme in only 3-month-old animals. The reduced IL-2 production of splenocytes as compared to younger animals and the activity in the mixed lymphocyte culture are also increased again in the old animals to the levels of the young mice (Hager, reference 113).
Extracts containing ganoderic acids and obtainable by extraction from shake cultures of G. lucidum after transfer of the mycelium into a stationary culture have anti-tumor activity (JP 4-304890 AA). It has proven particularly advantageous to extract a particular fraction from G. lucidum with water and others with ethanol and to combine both extracts (JP 0600222423 AA).
Extracts having antimicrobial activity: In vitro, from 0.01 to 1 mg/ml of a methanolic raw extract inhibit the formation of plaque caused by the caryogenic bacteria Streptococcus mutans (S. Hada, M. Hattori, T. Namba, Pharmacology 113 (1990), 73. An extract obtainable from the pileus of Ganoderma lucidum inhibits the growth of staphylococci (DE 693 18 921 T2). It is state of the art to combine extracts from G. lucidum with antibiotics; however, the antimicrobial activity cannot be predicted since antagonism also frequently occurs (Chemical Abstracts Vol. 131, Nos. 182 146).
In a concentration of 20 xcexcg/ml, a fraction of a methanolic extract from the fruit bodies referred to as F-III-4 and believed to contain ganoderic acids reduces the activity of reverse transcriptase in the culture supernatant of various HIV-infected cells by 40% and therefore deserves further interest as a potential anti-AIDS drug (Hager, reference 114). A triterpene obtainable by extraction from fungi of the Ganodermataceae, Polyporaceae or Lycoperdaceae families inhibits DNA polymerase (JP 0090-124690 AA).
Extracts having antiphlogistic activity: Aqueous extracts (2 g/kg, s.c.) exhibit a significant anti-inflammatory effect in the carrageen-induced ear edema of mice (Hager, reference 115). In the croton oil induced ear edema (mice), 500 xcexcg/ear of the ethyl acetate extract, upon topical application and after 6 hours, is as effective as 200 xcexcg/ear of hydrocortisone. Aqueous extracts (500 xcexcg/ear) have not been effective here. Six hours after p.o. administration (1 hour before the topical administration of croton oil), 500 mg/kg of the ethyl acetate or aqueous extract had as strong an anti-inflammatory effect as 18 mg/kg of hydrocortisone. The effect of the Ganoderma extracts lasted longer (24 hours) than that of hydrocortisone (Hager, reference 23). The antimicrobial effect of G. lucidum extracts has been used for impregnating textiles (JP 0110060424 AA). Also from other Basidiomycetes, e.g., Lentinus eodes, hot-water extracts which exhibited activity against HIV became obtainable after autolysis (DE 689 06 245 T2).
Extracts having radioprotective activity: Continuous i.p. administration of an aqueous extract of Ganoderma lucidum to 6 to 7 weeks old male ICR mice before and after exposure of the animals to X-rays (500 and 650 cGy) reduced the deleterious effects of the radiation and increased the number of surviving animals (Hager, reference 116).
The occurrence of hydroquinones in fungi of the genus Ganoderma has not been known to date. Inter alia, hydroquinones form part of the respiratory chain and of photosynthesis and are therefore wide-spread in nature. The free-radical scavenging property of hydroquinones is pharmacologically interesting and is utilized in medicaments.
For the total synthesis of the active substances of the hydroquinone type isolated from Ganoderma, the following prior art can be considered: The synthesis of [2,5-bis[(tert.-butyldimethylsilyl)oxy]phenyl]acetaldehyde required as an intermediate is effected by starting from 2,5-dihydroxyphenethyl alcohol according to D. G. Hahngauer (Tetrahedron Lett., 1986, 27, 5799-5802).
The synthesis of the terpenoid side chains recurred to L. Chen, G. B. Gill, G. Pattenden, H. Simonian (J. Chem. Soc., Perkin Trans. 1, 1996, 31-44), F. Kido, Y. Noda, T. Maruyama, C. Kabuto, A. Yoshikoshi (J. Org. Chem., 1981, 46, 4264-4266), M. A. Avery, M. S. Verlander, M. Goodman (J. Org. Chem., 1980, 45, 2750-2753), E. J. Corey, M. A. Tius, J. Das (J. Amer. Chem. Soc., 1980, 102, 1742-1744), E. J. Corey, A. Venktateswarin (J. Amer. Chem. Soc., 1972, 94, 6190-6191), J. A. Marshall, D. G. Cleary (J. Org. Chem., 1986, 51, 858-863), T. R. Hoye, M. J. Kurth (J. Org. Chem., 1980, 45, 3549-3554), G. Beck, D. Gunther (Chem. Ber., 1973, 106, 2758-2766), and D. Grassi, V. Lippuner, M. Aebi, J. Brunner, A. Vasella (J. Amer. Chem. Soc., 1997, 119, 10992-10999).
The prior art further includes the oxidation of hydroquinones to quinones, which can be effected with various oxidants (W. M. Owton, J. Chem. Soc., Perkin Trans. 1, 1999, 2409-2420). Further, it is possible to employ 4-substituted phenols as a starting material. In the ruthenium-catalyzed oxidation with tert.-butyl hydro-peroxide in ethyl acetate, a rearrangement occurs to obtain 2-substituted quinones (W. M. Owton, J. Chem. Soc., Perkin Trans. 1, 1999, 2409-2420). Effectiveness against multiresistant bacteria has been known of none of the hydroquinones and quinones prepared to date by various methods. The same applies to conversion products of such hydroquinones and quinones known to date.
Mono- and dihydroquinone ethers are derived from hydroquinone by alkylation or arylation. In the medicine, hydroquinone benzyl ethers are employed against hyperpigmentations of the skin (liver spots, freckles) (Rompp Chemie-Lexikon, Georg Thieme Publishers Stuttgart, New York, 9th Edition, 1995). From the glucoside of hydroquinone, arbutin, which occurs, inter alia, in the leaves of Arctostaphylos uva-ursi (Ericaceae, bearberry), hydroquinone is released in the organism under certain conditions. The latter is held responsible for the urine-disinfecting activity of the drug (E. Teuscher, Biogene Arzneimittel, Wiss. Verlagsgesellschaft mbH, Stuttgart 1997). A coupling of active substances obtainable from Ganoderma species with other antibiotics has not been described to date. In principle, coupling reactions on antibiotics aiming at enhancing the effect by chemical conversions are known. Esterification of ofloxacin with ethanol is already known: J. S. Kiely, E. Laborde, L. E. Lesheski, R. A. Busch (J. Heterocycl. Chem., 1991, 28, 541-543).
Esterifications of penicillin G with simple alcohols also belong to the state of the art, e.g.: M. Murakami, M. Hajima, F. Takami, M. Yoshioka (Heterocycles, 1990, 31, 2055-264). The state of the art involves the use of enzymes to enable conversion under mild conditions.
The synthesis of cephalosporins is effected, for example, by the acylation of 7-aminocephalosporinic acid and 7-aminodeacetoxycephalosporinic acid by the catalytic action of acylases from B. megaterium or E. coli strains (T. Fujii, K. Hanamitsu, R. Izumi, T. Yamaguchi and T. Watanabe (1973), Japanese Patent 7399393, SNAM, Proetti (1972), Belgian Patent 782646). Lipases catalyze the esterification of a broad range of substrates (Ching et al., Angew. Chem. 101 (1989), 711-724). Therefore, the derivatization of new active substances from Ganoderma species can rely on these synthetic principles of the prior art in order to arrive at basically novel hybrid antibiotics.
The occurrence of Ganoderma pfeifferi as a tree fungus has long been known. However, no biologically active compounds trom the fungus Ganoderma pfeifferi have been known to date. Extracts from G. pfeifferi have not been reported in the literature, let alone employed as medicaments. The cultivation of Ganoderma pfeifferi in mushroom farms or in laboratory cultures has not been reported previously.
Although it could be expected that other Ganoderma species contain similar compounds as those isolated from G. lucidum, in contrast to the intensive medicinal utilization of G. applanatum and especially of G. lucidum, a utilization of other Ganoderma species and especially of G. pfeifferi has not been possible to date. Thus, the fungus is publicly accessible, but a commercial utilization of the wild fungi has not been effected previously. Thus, the valuable potential of this fungus has remained unused to date.
Antibiotics include substances produced by fungi and derivatives formed thereof which are able to control infections upon intrasomal application.
The components of G. lucidum and G. applanatum have not been used for the development of antibiotics although the antimicrobial activity of some extracts has been known. This is due to the fact that the various activities of the extracts from G. lucidum are very difficult to attribute to a specific active substance. It is just the wide variety of active substances from G. lucidum mentioned in the prior art which proves to be an obstacle to the development of an antibiotic because a substance having a dominant antimicrobial activity for which the recovery as an individual substance or the total synthesis appeared to be promising has not been isolated from G. lucidum and G. applanatum. The active substances isolated to date from G. lucidum or G. applanatum do not exhibit sufficient antimicrobial activity as individual substances. The extracts are difficult to standardize (JP 0610225649 AA), indefinite in their antimicrobial activity and therefore unsuitable as antibiotics.
However, the current state of the demands imposed on an antibiotic requires a well characterized active substance with defined pharmacokinetics and reproducible antimicrobial properties for the use as an antibiotic. To date, this precondition was not met by the materials recovered from G. lucidum or G. applanatum. Despite of the intensive search for active substances from fungi of the genus Ganoderma which can be used for pharmaceutical purposes, the isolation of compounds which are promising for the development of antibiotics due to their properties, the preparation of such active substances by chemical synthesis and derivatization have not been successful. Although a total synthesis of an active substance isolated from fungi is not indispensable to the development of an antibiotic, the preconditions for the development of an antibiotic are essentially improved by solving this problem.
Due to the increasing problems with resistances, there is still a high demand for antibiotics which is not satisfied by the extracts and active substances described in the prior art.
Especially in severe Gram-positive infections, only glycopeptide antibiotics are still sufficiently effective currently. However, staphylococci and enterococci are increasingly developing resistances against antibiotics from this group of substances, but also Gram-negative pathogens, for example, of the genus Pseudomonas, increasingly develop multiresistances so that infections with Gram-negative pathogens will no longer be safely controllable clinically in the future either. Also in veterinary medicine, infections of useful animals with multiresistant staphylococci become increasingly threatening. Additional danger is impending from a possible transfer to humans of pathogens having become resistant. In addition, the problem of infection control has been solved only unsatisfactorily to date in many other fields, e.g., in fish breeding. The increasing spread of breeding systems has led to great difficulties. Due to the ecological problems, only a few agents have been approved to date for treatment in fish-breeding systems. The problems cannot be solved with the active substances described in the prior art.
To overcome the problems of resistance in human and veterinary medicine, it is urgently necessary to develop new antibiotics. The object to be solved by the invention has been to make further Ganoderma species utilizable for medicinal use, to recover extracts therefrom, and to isolate active substances from such extracts and to further develop them by chemical conversions. In particular, it has been the object of the invention to meet the demand for new antibiotics due to the increasing development of bacterial resistances against conventional antibiotics, especially in the therapy of infectious diseases in human and veterinary medicine, by providing as yet unknown active substances from the genus Ganoderma and derivatives thereof.
To improve the preconditions for the development of an antibiotic, solutions should be found for also preparing the isolated active substances and products derived therefrom by chemical synthesis.
The object has been achieved by the recovery of as yet unknown active substances from naturally occurring fungi of the species G. pfeifferi, the culturing of G. pfeifferi DSM 13239 in mushroom farms, the culturing of G. pfeifferi DSM 13239 in fermenters, the preparation of different extracts from the cultured fungus, the isolation of antimicrobially active substances from these extracts, the preparation of these active substances by total synthesis, and the derivatization of these active substances.
The fungus of the species G. pfeifferi was deposited on Jan. 11, 2000, with the international deposit center DSMZ, Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, in accordance with the regulations of the Budapest Treaty. The deposit number is DSM 13239.
According to the invention, biologically active extracts from Ganoderma pfeifferi DSM 13239 can be obtained by treatment with solvents.
The extracts according to the invention can be obtained from both the fruit body of naturally occurring fungi of the species Ganoderma pfeifferi and the fruit bodies of fungi grown in mushroom farms.
The extracts can be advantageously obtained from the fruit body of the species Ganoderma pfeifferi DSM 13239 if the fungi are grown on wood substrates in mushroom farms. An as yet not realized technical solution for the medicinal utilization of the valuable components of G. pfeifferi is thereby achieved.
In a particularly advantageous way, the extracts can be obtained from the fruit body of the species Ganoderma pfeifferi DSM 13239 when the culturing of the fungi was performed after pretreating the wood with cellulose-degrading enzymes.
The extracts according to the invention can be obtained from the mycelium of the species Ganoderma pfeifferi DSM 13239 after growth in fermenters. Thus, another technical solution for the recovery of the components of Ganoderma species, especially Ganoderma pfeifferi DSM 13239, is achieved. Extracts obtainable from the mycelium of the species Ganoderma pfeifferi DSM 13239 after growth in fermenters are preferably employed since this opens a possibility for the recovery of biologically effective extracts and compounds which is independent of the tedious production of fruit bodies. It has proven advantageous to add ammonium succinate to the liquid medium. The extracts according to the invention can be obtained from the mycelium of the species Ganoderma pfeifferi DSM 13239 wherein the fungi are cultured in liquid media with carbohydrate sources, preferably in malt medium having a content of malt extract of from 20 to 40 g/1000 l and an initial pH of 4.5-7.5, with observance of a lighting and shaking regimen. In particular, it is advantageous to add wood extract, especially decoction from beech, alone or in combination with cellulose-degrading enzymes to the liquid medium.
From the fruit body and/or mycelium of the naturally occurring fungus or of the fungus grown by this method, extracts can be obtained with solvents of different polarities. Known extractants, such as cold and hot water, methanol, ethanol, acetone, ethyl ether, can be employed, but solvents which have not previously been employed in the extraction of Ganoderma species, such as dichloromethane and ethyl acetate, are particularly advantageous. Extracts according to the invention are advantageously obtained by extracting the fruit body or the mycelium with a lipophilic solvent, especially dichloromethane. Extracts according to the invention are also obtained by extracting the fruit body and/or the culture medium and/or the mycelium with ethyl acetate. Extracts obtainable by extraction with monohydric alcohols are recovered in particularly high yields. In this case too, the addition of wood decoction according to the invention is beneficial.
Also, it is in accordance with the invention to obtain extracts by water extraction, preferably at temperatures of from 10xc2x0 C. to 80xc2x0 C., wherein the extraction is advantageously effected stepwise with water of increasing temperature.
It is possible to perform the extraction in several steps with solvents having different polarities. Extracts according to the invention are obtained by extracting the residue obtained in the extraction with lipophilic solvents.
Extracts obtained by using monohydric alcohols for extracting the residue of dichloromethane extraction are advantageous.
Extracts obtained by using monohydric alcohols for extracting the residue of dichloromethane extraction can be further purified in a particularly advantageous way by partitioning with ethyl acetate and further purification on silica gel using gradients with the solvent dichloromethane/ethyl acetate 4:1 or on Sephadex. Extracts obtained by a water extraction of the residue of dichloromethane extraction and/or of the residue of ethanol extraction are also in accordance with the invention.
Extracts obtainable from the fruit bodies of naturally occurring fungi or of fungi grown in mushroom farms and from the mycelium of the species Ganoderma pfeifferi DKMS after growth in fermenters have proven to be biologically active. Surprisingly, ethanolic and aqueous extracts from the mycelium have a strong antiviral effect. With lipophilic extractants, extracts are obtained which exhibit effectiveness against Gram-positive bacteria. Monohydric alcohol extraction of the fruit bodies or mycelium of the strains of G. pfeifferi DSM 13239 grown according to the invention yields extracts which are effective against Gram-negative rod bacteria. Alcoholic extracts exhibit particular effectiveness against multiresistant bacteria of the genus Pseudomonas, which is not achieved with the dichloromethane extracts. Particularly active extracts are obtained by extracting with ethyl acetate the fruit body and/or the culture medium and/or the mycelium and/or the residue of extractions. Advantageously, the residue of extractions with monohydric alcohols can be employed. By another fractionation with a dichloromethane/ethyl acetate gradient, fractions having a high antimicrobial activity can be obtained.
It could not be predicted that an additional extract capable of inhibiting bacteria could be respectively obtained, for example, from the residue of dichloromethane extraction by ethanolic extraction, and from the residue of dichloromethane extraction and ethanolic extraction by a hot-water extraction.
In different fields of application, the extracts can be directly employed. It is also in accordance with the invention to use chromatographic methods for further processing the extracts obtainable from the fruit body or mycelium of strains of G. pfeifferi DSM 13239 cultured according to the invention, and to isolate pure active substances therefrom.
As expected, active substances known from G. lucidum or G. applanatum, such as ganoderol B, applanoxidinic acid G or triterpenes, can also be obtained thereby. Surprisingly, however, further active substances, such as the as yet unknown triterpene 3,26-dihydroxylanosta-8,24-diene-7-one, which we have called ganoderone B, can additionally be isolated. Triterpenes of the composition 3,26-dihydroxylanosta-8,24-diene-7-one can be obtained from the extracts of the fruit body and mycelium of G. pfeifferi DSM 13239 by extraction with solvent and have a remarkable biological activity. Ganoderone B has antiviral activity against influenza virus type A and inhibits the lipopolysaccharide binding to CD14+ cells.
Within the scope of the various pharmacological activities of the components of G. lucidum and G. applanatum, antibacterially active substances have as yet played an inferior role. Therefore, it could not be foreseen that, from the wide variety of possible extracts from G. pfeifferi DSM 13239, especially from the fruit body, there were prepared extracts having an antimicrobial activity which has a clearly superior activity as compared to that of extracts from G. lucidum. It was totally surprising that biologically active substances of general formula 1 can be obtained which have not previously been described either in other Ganoderma species or in other fungi, plants or animals. The compounds of general formula 1, referred to as ganomycins, are suitable as guiding structures for the development of antibiotics.
Active substances of formula 1 with residues R1-R5 can be obtained by the growth and extraction methods developed by us, wherein R1, R2 and R3 represent hydrogen, R5 represents CH3 and CH2OH, and R4 is in the form of a free acid; these active substances can also be obtained by chemical synthesis, which yields derivatives in which R1, R2 and R3 represent hydrogen, halogen, alkyl or alkoxy groups, R5 represents a xe2x80x94CH2xe2x80x94aryl, xe2x80x94CH2xe2x80x94alkyl, xe2x80x94CH2-O-aryl, xe2x80x94CH2xe2x80x94O-alkyl, xe2x80x94CH2N3, xe2x80x94CH2-carboxylic acid, xe2x80x94CH2-aldehyde or xe2x80x94CH2-alcohol group, xe2x80x94CH2NR92, xe2x80x94CX3, xe2x80x94CHX2, xe2x80x94CH2X or xe2x80x94CH3, or bound to a carbohydrate derivative through a heteroatom, wherein X=F, Cl, Br, and R9 is a hydrogen atom, an alkyl or aryl residue, and R4 is selected to yield the free acid, an acid halide, amide, a salt or an ester with aliphatic or aromatic alcohols of the compound, and n is a number of from 1 to 10.
As stated in the Drawbacks of the Prior Art, it is a particular advantage to the development of an antibiotic when the natural substance found can also be prepared by a total synthesis.
Active substances which are provided with the following substituents in formula 1: R1, R2 and R3=H, F, Cl, Br, I or O-n-alkyl, R4=MeI, MeII, ammonium, alkylammonium, aryl or alkyl, R5=H, alkyl, aryl, Rxe2x80x2OH, CHO, Rxe2x80x2CHO, COOH or Rxe2x80x2COOH (Rxe2x80x2=aryl or alkyl); can be obtained by chemical synthesis by varying the synthetic route and the starting components.
According to the invention, this object was achieved by a variety of chemical processes.
By using substituted (2,5-dihydroxyphenyl)acetaldehydes, the found antimicrobially active basic structure can be further modified on positions R1 to R3. A particular amplification of activity is achieved by introducing halogens, especially fluorine, and of (2,5-dihydroxyphenyl)acetaldehydes substituted with methoxy groups on the ring system.
A process for the preparation of novel substituted hydroquinones and their derivatives is characterized in that the coupling of the hydroquinone substituted with residues R1-R3 to the terpenoid side chain is effected by starting from halogen-substituted hydroquinones via organometallic intermediates through the reaction with a terminal epoxide function of the terpenoid side chain.
It is also possible to effect the preparation of novel substituted hydroquinones and their derivatives in such a way that the coupling of the hydroquinone substituted with residues R1-R3 to the terpenoid side chain is effected by starting from halomethyl-substituted hydroquinones via organometallic intermediates through the reaction with a carbonyl function of the terpenoid side chain.
Another possibility for the preparation of novel substituted hydroquinones and their derivatives is characterized in that the coupling of the hydroquinone substituted with residues R1-R3 to the terpenoid side chain is effected by starting from substituted (2,5-dihydroxyphenyl)acetaldehydes through reaction with carbanions intermediately produced from suitable terpenoids.
The preparation of novel substituted hydroquinones and their derivatives may also be effected in such a way that the coupling of the hydroquinone substituted with residues R1-R3 to the terpenoid side chain is effected by starting from substituted 4-(2,5-dihydroxyphenyl)-2-methylidenecarboxylic acid esters bearing an electron-withdrawing leaving group in position 3 through reaction with carbanions intermediately produced from suitable terpenoids.
The process for the preparation of novel substituted hydroquinones and their derivatives is characterized by the stepwise deprotection of the synthesized compounds in order to obtain the free active substance or perform further functionalizations.
With different xcex1-sulfonated carbonyl compounds, different side chains can be realized.
By alkylation or arylation of the active substances according to formula 1, mono-(formula 2) and dihydroquinone ethers (formula 3) can be obtained, wherein R6, R7 can be aryl, alkyl. These are also biologically active.
By introducing acid anhydrides as substituents on the carbon atom 18, biologically active compounds having a higher hydrophilicity are obtainable. Reaction with phthalic acid anhydride yields the active substance according to formula 4. By oxidation of the hydroquinones according to formula 1, the corresponding biologically active quinones (formula 5) can be obtained.
Active substances of general formula 1 can be linked by esterification to antibiotics bearing acid groups. On the one hand, this may be done on a chemical route using known reactions. The core of the present invention is the combination of new and known elements, whereby the choice of antimicrobially active compounds which are suitable for the treatment of infections with multiresistant germs is substantially enlarged. The wide variety of possible reactions for the active substances of formula 1 opens totally new routes in antibiotic therapy. According to the invention, it is particularly advantageous to effect the esterification by biotransformation. Novel antimicrobially active substances of formulas 5 to 12 and processes for their preparation are characterized in that active substances of general formula 1 are linked to antibiotics bearing amino groups through coupling at carbon atom 11. It is preferably to use active substances of general formula 1 with R4=CH3 which are linked to antibiotics bearing amino groups through coupling at carbon atom 11, because the yields can be increased by 20% thereby.
By esterification of the hydroquinones of formula 1 (R5=CH2OH), obtainable from G. pfeifferi DSM 13239 or prepared synthetically, with cis-1,2-epoxypropyl-phosphonic acid (fosfomycin), the active substance according to formula 6 can be obtained which will be bound particularly strongly to the surface of staphylococci through bonding to UDP N-acetyl-D-glucosaminyl-3-enol pyrovyl transferase. Thus, on the one hand, the synthesis of the bacterial cell wall is disturbed, and on the other hand, due to the particular properties of hydroquinone, the redox potential in the environment of the cell is influenced and the activity of extracellular enzymes is thus reduced.
Coupling the hydroquinones of formula 1 (R5=CH2OH), obtainable from G. pfeifferi DSM 13239 or prepared synthetically, with the synthetically preparable inhibitor of cell wall synthesis, fosfonochlorine, yields an active substance of formula 7 which causes a particularly intense disturbance of the bacterial cell wall.
Binding the hydroquinones of formula 1 (R5=CH2OH), obtainable from G. pfeifferi DSM 13239 or prepared synthetically, to liposidomycin yields an active substance of formula 8 which can be used to selectively influence the bacterial peptidoglucane synthesis.
Esterification of the hydroquinones of formula 1 (R5=CH2OH), obtainable from G. pfeifferi DSM 13239 or prepared synthetically, with cephalosporin C results in an active substance of formula 9. The active substances of formula 10 obtained by reaction with xcex2-lactam antibiotics inactivate the bacterial penicillinases and partially offset existing resistances. If the resistance occurred because the bacterial cell formed receptors having a particularly high affinity for penicillin, formation of the hydroquinones on the bacterial cell wall is achieved. Therefore, the hydroquinones can display their enzyme-inhibiting properties in immediate vicinity of the bacterial cell. Since Gram-positive bacteria rely on the activity of extracellular enzymes, an antibacterial effect is thus achieved.
By esterification of the hydroquinones of formula 1 (R5=CH2OH), obtainable from G. pfeifferi DSM 13239 or prepared synthetically, with fusidinic acid, the active substance 11 can be obtained. This reaction causes an increase of surface activity and an enhancement of lipophilic properties.
Esterification of the active substances of formula 1, R5=CH2OH, with ofloxacin results in active substance 12 by means of which an enlargement of the range of activities can be achieved.
Further novel antimicrobially active substances of formula 1 are obtained by binding to carbohydrate derivatives through a heteroatom at C18. The reaction of methyl-2,3,4-tri-O-benzyl-6-deoxy-6-iodo-xcex2-D-glucopyranoside yields 11-(methyl-2,3,4-tri-O-benzyl-6-deoxy-xcex2-D-glucopyranos-6-yl)oxy(2(1xe2x80x2)Z,5E,9E)-2-[2xe2x80x2-(2,5-dihydroxyphenyl)ethylidene]-6,10-dimethyl-5,9-undecadienic acid which interacts with the bacterial cell membrane due to its carbohydrate moiety.
Therefore, it is possible to adapt the properties of the ganomycins of general formula 1 to the intended use by biotransformation and/or chemical derivatization.
The use of one or more of compounds of formula 1 and/or extracts according to Example 1 as free-radical scavengers is possible both in preservation technology and in various diseases. Also, the use of one or more of compounds of formula 1 and/or extracts according to claim 1 for inhibiting the activity of neutral endopeptidases and/or the angiotensin-converting enzyme is possible in various diseases because the proteases participate in numerous posttranslational processes of the functional control of mammal organisms. By inhibiting these enzymes, anti-inflammatory, immunostimulant, analgetic, antihypertensive and antiviral activities have been achieved.
The use of one or more of compounds of formula 1 and/or extracts according to Example 1 for inhibiting the serum-mediated binding of lipopolysaccharides causes fever reduction and normalization of the distension of peripheral blood vessels in certain clinical pictures.
The use of biologically active extracts and/or compounds from fungi of the genus Ganoderma as substances and extracts having antimicrobial activity as a preservative for technical purposes, in particular, predominantly utilizes the antimicrobial properties.
The use of biologically active extracts and/or compounds from fungi of the genus Ganoderma as substances and extracts used as additives for pharmaceutical and cosmetic formulations, especially as preservatives, utilizes the favorable combination of antimicrobial activity, free-radical scavenging property, inhibition of proteases and the resulting anti-inflammatory effect.
The use of biologically active extracts and/or compounds from fungi of the genus Ganoderma as vitalizing and germ-reducing additives for foods, as food supplements and health-care agents also utilizes the combination of favorable properties and additionally causes relief of chronic pain.
The use of biologically active extracts and/or compounds from fungi of the genus Ganoderma for application in human and veterinary medicine, especially in the control of infections, as a sole active substance and in the form of combination preparations solves problems recently arisen in the control of multiresistant bacteria. In human medicine, the control of severe Gram-positive infections, which can be lethal as a consequence of sepsis, is becoming increasingly more difficult. Therefore, it is a particular advantage to be able to choose from a broad range of antimicrobially active compounds. The enlargement of the range of antimicrobially active substances by derivatizing the active substances according to formula 1 is therefore very important to provide agents having antimicrobial activity and different application properties for human and veterinary medicine. This applies, in particular, to a use with multiresistant Gram-positive germs. In veterinary medicine, staphylococcus infections play a great role mainly in the keeping of cattle because the germs are easily communicated during milking and can contaminate the milk. The application of the active substances of formula 1 can be done both systemically and locally.
The use as an agent against fish-pathogenic bacteria and in fish breeding results from the established activity against fish-pathogenic germs.
A particular advantage which results from the above properties is the use as agents with antimicrobial activity and as a free-radical scavenger. This use is characterized in that the extracts and the compounds isolated therefrom are used alone and in mutual combination. The established biological activities underlie the inventive application as a medicament containing one or more of the compounds and/or extracts. Formulations containing one or more of the compounds and/or extracts can be employed, in particular, for the preparation of a medicament for the treatment of Gram-positive infections and the sepsis caused by the infection. From the specific inhibiting activity of the extracts from G. pfeifferi DSM 13239 towards neutral endopeptidase, a use in the treatment of pain can be derived by interfering with the degradation of opinoid peptides and endorphins which act on the opiate receptors. On the other hand, inhibition of the neutral endopeptidase causes an inhibition of the degradation, suppression of aldosterone synthesis and enhancement of the renal sodium excretion. Thus, extracts from G. pfeifferi DSM 13239 can be employed in the treatment of hypertension. The use of one or more of the compounds and/or extracts obtainable from G. pfeifferi DSM 13239 for inhibiting the activity of neutral endopeptidases results in a substantial extension of the possible applications. Therefore, the use of one or more of the compounds and/or extracts according to the invention for the preparation of a medicament for the treatment of hypertension, cardiovascular diseases and metabolic disorders is possible.